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- Title
Establishment of a medium-throughput approach for the genotyping of RHD variants and report of nine novel rare alleles.
- Authors
Fichou, Yann; Le Maréchal, Cédric; Jamet, Déborah; Bryckaert, Laurence; Ka, Chandran; Audrézet, Marie ‐ Pierre; Le Gac, Gérald; Dupont, Isabelle; Chen, Jian ‐ Min; Férec, Claude
- Abstract
Background The routinely used serologic methods are robust in accurately typing standard D− or D+ blood. However, they result in discrepancy in weak or partial D blood, which requires genetic analysis. We have previously used denaturing high-performance liquid chromatography ( DHPLC) to screen the entire RHD-coding sequence. However, DHPLC is technically challenging, labor-intensive, and time-consuming. To overcome these inconveniences, we sought to develop a new two-step approach. Study Design and Methods A total of 430 blood samples with D phenotype ambiguity were recruited for this study. The three most frequent weak D alleles (i.e., weak D, Type 1; weak D, Type 2; and weak D, Type 3), which altogether account for 60% to 90% of the atypical RHD alleles in the Caucasian population, were first identified by Tm-shift genotyping. The remaining unidentified samples were then subjected to a single-tube multiplex polymerase chain reaction ( PCR) amplification of all 10 RHD exons followed by direct sequencing. Results Optimal conditions for efficient and reliable identification of the three most common weak D variants by Tm-shift genotyping were established. All 10 RHD exons were successfully amplified in a single-multiplex PCR procedure. Employment of the two-step analysis identified RHD variants in 91.6% of the 430 studied samples. Two of the nine previously undescribed variants, c.335 G> T and c.939 G> A, were found to cause aberrant mRNA splicing by means of a splicing minigene assay. Conclusion The new two-step analysis proved to be much easier and cheaper than the DHPLC method and therefore is convenient to be used as a routine, medium-throughput approach for RHD genotyping.
- Subjects
RABBIT calicivirus disease; ALLELES; SERODIAGNOSIS; HIGH performance liquid chromatography; BLOOD sampling; HUMAN phenotype
- Publication
Transfusion, 2013, Vol 53, Issue 8, p1821
- ISSN
0041-1132
- Publication type
Article
- DOI
10.1111/trf.12009